The present invention relates to an enzyme having carbonyl reduction activity of reducing a carbonyl compound asymmetrically to produce an optically active alcohol (hereafter, such an enzyme is referred to as a CRD enzyme), a DNA coding such an enzyme, a plasmid having such a DNA, a transformant which is a cell transformed with such a plasmid, and a production method of an optically active alcohol using the enzyme and/or the transformed cell. The resultant optically active alcohol, for example, (S)-4-halo-3-hydroxy butyric ester, is a useful compound as a raw material for the synthesis of medicines, agricultural chemicals, and the like.
A number of CRD enzymes are known (see Yuki Gosei Kagaku, 49, 52 (1991) and Eur. J. Biochem., 184, 1 (1981)). Among such CRD enzymes, those which act on 4-halo acetoacetic ester to produce (S)-4-halo-3-hydroxy butyric ester, which are derived from microbes, and which have reported characteristics, are only a Geotrichum candidum derived enzyme (Enzyme Microb. Technol. (1992), Vol. 14, 731) and a Candida parapsilosis derived enzyme (Enzyme Microb. Technol. (1993), Vol. 15, 950). However, no information has been reported on genes coding these two types of enzymes. The reduction of 4-halo acetoacetic ester using such enzymes only proceeds at a low substrate concentration. It is therefore impractical to synthesize (S)-4-halo-3-hydroxy butyric ester using such enzymes as catalysts.
Besides the above reaction using the two types of enzymes, a number of reactions using microbe bodies and the products of such reactions are known to realize asymmetric reduction of 4-halo acetoacetic ester (Japanese Patent No. 1723728, Japanese Laid-Open Publication Nos. 6-209782 and 6-38776, etc.) However, such reactions are not performed at a high substrate concentration, and thus it cannot be asserted that a practical production method has been established. See, for example, a reaction method using a two-phase system with an organic solvent (Japanese Patent No. 2566962). A method using a ruthenium-optically active phosphine complex as a catalyst has also been reported (Japanese Laid-Open Publication No. 1-211551). This method however has many problems to be solved, such as the requirement of a high-pressure reaction vessel and need for an expensive catalyst.
Under the above circumstances, development of a practical enzyme has been desired for use in asymmetric reduction of a carbonyl compound such as 4-halo acetoacetic ester to produce an optically active alcohol such as (S)-4-halo-3-hydroxy butyric ester.
A CRD enzyme requires a reduction-type coenzyme for reaction. Conventionally, when a carbonyl compound is to be reduced using a microbe body and the like having a CRD enzyme, a saccharide such as glucose is added to a reaction system to activate a group of regeneration-system enzymes for changing an oxidized coenzyme to a reduced type, thereby regenerating the coenzyme so as to be used for the reduction. Such a group of regeneration-system enzymes are likely to be blocked or damaged by substrates and reduced products. This has been considered to be one of major reasons why the reduction proceeds only when the concentration of substrates or products is low. It is known that the amount of an expensive coenzyme used during reduction can be greatly reduced by combining an enzyme having- the ability of regenerating a coenzyme on which a CRD enzyme depends with the CRD enzyme during the reaction (Japanese Patent No. 2566960 and Enzyme Microb. Technol. (1993), Vol. 15, 950, for example). In this case, however, it is required to prepare an enzyme source for regenerating the coenzyme separately from the preparation of the CRD enzyme before the regenerating enzyme is added to a reaction system.
The Inventors of the present application have discovered a novel Candida-genus derived CRD enzyme, and found that an optically active alcohol can be efficiently produced from a carbonyl compound by using this CRD enzyme.
Also found is that an optically active alcohol can be efficiently produced by using a transformed cell containing a gene of an enzyme having the ability of regenerating a coenzyme (e.g., a glucose dehydrogenase gene) concurrently.
Thus, the present invention to be described in the specification can advantageously provide a novel CRD enzyme, a DNA coding this enzyme, a plasmid having this DNA, a transformant which is a cell transformed with this plasmid, and a production method of an optically active alcohol using the above enzyme and/or transformed cell.
The carbonyl reductase according to the present invention has physical and chemical properties (1) to (4) of:
(1) action:
acting on ethyl 4-chloroacetoacetate using NADPH as a coenzyme to produce ethyl (S)-4-chloro-3-hydroxybutyrate;
(2) substrate specificity:
exhibiting a strong activity to ethyl 4-chloroacetoacetate while exhibiting substantially no activity to ethyl acetoacetate;
(3) optimal pH: 5.5 to 6.5; and
(4) action optimal temperature: 50xc2x0 C. to 55xc2x0 C.
In one embodiment, the carbonyl reductase has additional physical and chemical properties (5) to (7) of:
(5) heat stability: being stable up to about 40xc2x0 C. when processed at pH 7.0 for 30 minutes;
(6) inhibitor: being inhibited by mercury ions and quercetin; and
(7) molecular weight: about 76,000 by gel filtration analysis and about 32,000 by SDS polyacrylamide electrophoresis analysis.
The carbonyl reductase according to the present invention has an amino acid sequence of SEQ ID NO:1 of the Sequence Listing or an amino acid sequence with one or several amino acids being deleted, substituted, or added in the amino acid sequence of SEQ ID NO:1 of the Sequence Listing, or part of the amino acid sequences of SEQ ID NO:1 of the Sequence Listing, and having an activity of reducing ethyl 4-chloroacetoacetate asymmetrically to produce ethyl (S)-4-chloro-3-hydroxybutyrate.
In one embodiment, the enzyme is obtained from a microbe belonging to genus Candida. In a preferred embodiment, the enzyme is obtained from Candida magnoliae. In a more preferred embodiment, the enzyme is obtained from Candida magnoliae IFO 0705.
The DNA according to the present invention codes for the above enzyme. In one embodiment, the DNA has a nucleotide sequence of SEQ ID NO:2 of the Sequence Listing.
The plasmid according to the present invention has the above DNA sequence. In one embodiment, the plasmid is pNTS1.
The transformed cell according to the present invention is a transformant which is a cell transformed with the above plasmid. In one embodiment, the transformed cell is E. coli. In a preferred embodiment, the transformed cell is E.coli HB101 (pNTS1).
The plasmid according to the present invention has a DNA coding for an enzyme having an activity of asymmetrically reducing ethyl 4-chloroacetoacetate to produce ethyl (S)-4-chloro-3-hydroxybutyrate and a DNA coding for an enzyme having an ability of regenerating a coenzyme on which the enzyme depends (e.g., glucose dehydrogenase).
In one embodiment, the glucose dehydrogenase is derived from Bacillus megaterium. In a preferred embodiment, the plasmid is pNTS1G.
The transformed cell according to the present invention is a transformant which is a cell transformed with the above plasmid.
In one embodiment, the transformed cell is E.coli. In a preferred embodiment, the transformed cell is E.coli HB101 (pNTS1).
The transformed cell according to the present invention is a transformant which is a cell transformed with a first plasmid having a DNA coding for an enzyme having an activity of asymmetrically reducing ethyl 4-chloroacetoacetate to produce ethyl (S)-4-chloro-3-hydroxybutyrate, and a second plasmid having a DNA coding an enzyme having an ability of regenerating a coenzyme on which the enzyme depends (e.g., glucose dehydrogenase).
In one embodiment, the transformed cell is a transformant which is a cell transformed with plasmid pNTS1 and a plasmid having a DNA coding for glucose dehydrogenase derived from Bacillus megaterium. In a preferred embodiment, the transformed cell is E. coli. 
The production method for producing an optically active 3-hydroxy butyric ester according to the present invention includes the steps of: reacting with a 3-oxo-butyric ester an enzyme having an activity of asymmetrically reducing a 3-oxo-butyric ester to produce an optically active 3-hydroxy-butyric ester or a culture of a microbe having an ability of producing the enzyme or a processed product of the culture; and harvesting a produced optically active 3-hydroxy-butyric ester.
The production method for producing an optically active 3-hydroxy butyric ester according to the present invention includes the steps of: reacting a transformant which is a cell transformed with a plasmid having a DNA coding for an enzyme having an activity of asymmetrically reducing a 3-oxo-butyric ester to produce an optically active 3-hydroxy-butyric ester with a 3-oxo-butyric ester: and harvesting a produced optically active 3-hydroxy-butyric ester.
The production method for producing an optically active alcohol according to the present invention includes the steps of: reacting with a carbonyl compound a transformant which is a cell transformed with a plasmid having a DNA coding for an enzyme having an activity of asymmetrically reducing a carbonyl compound to produce an optically active alcohol and a DNA coding an enzyme having an ability of regenerating a coenzyme on which the enzyme depends; and harvesting a produced optically active alcohol.
The production method of an optically active alcohol according to the present invention includes the steps of: reacting with a carbonyl compound a transformant which is a cell transformed with a first plasmid having a DNA coding for an enzyme having an activity of asymmetrically reducing a carbonyl compound to produce an optically active alcohol and a second plasmid having a DNA coding for an enzyme having an ability of regenerating a coenzyme on which the enzyme depends; and harvesting a produced optically active alcohol.
In one embodiment, the carbonyl compound is a 3-oxo-butyric ester represented by a general formula: 
and the resultant optically active alcohol is an optically active 3-hydroxy-butyric ester represented by a general formula: 
In a preferred embodiment, in the above general formulae, R1 and R2 are independently halogen, azide, benzyl amino, or hydrogen, one of R1 and R2 being hydrogen, and R3 is a substituted or non-substituted alkyl group or aryl group.
In a more preferred embodiment, in the above general formulae, R1 is chlorine, R2 is hydrogen, and R3 is ethyl.
In a preferred embodiment, in the above general formulae, R1 and R2 are independently an alkyl group, a hydroxyl group, or hydrogen, one of R1 and R2 being hydrogen, and R3 is a substituted or non-substituted alkyl group or aryl group.
In a more preferred embodiment, in the above general formulae, R1 is a hydroxyl group, R2 is hydrogen, and R3 is ethyl.